This invention relates to a culture vessel for cell cultures having at least one cell culture chamber containing a cell culture mixture, which is separated by a dialysis membrane from a nutrient chamber. Nutrients are transported from the nutrient chamber through the dialysis membrane into the cell culture chamber and metabolic products are transported out of the cell culture chamber into the nutrient chamber. A feed and discharge system is provided for the gases required and generated during cell culturing.
Culture vessels of this kind can be used, for example, for in vitro production of monoclonal antibodies. Monoclonal antibodies are currently produced for a number of purposes in diagnosis, treatment, and biomedical research, usually using hybridoma technology methods. "Hybridoma cells" is the term for immortalized hybrids of antibody-producing cells and myeloma cells. The antibodies produced by the hybridoma cells, which are characterized by high specificity, are referred to as "monoclonal" antibodies.
For in vitro antibody production, these hybridoma cells are cultured in certain liquid media whose composition corresponds in many respects to that of blood. These media contain ingredients which include salts, sugar, vitamins, amino acids, and a buffer system based in most cases on sodium hydrogencarbonate (NaHCO.sub.3). Usually the hybridoma cells are cultured in an incubator atmosphere with high atmospheric humidity and a CO.sub.2 content that is at equilibrium with the NaHCO.sub.3 present in the medium.
The monoclonal antibodies produced in this matter with conventional stationary in vitro methods, in the form of tissue culture products, are very well suited for many purposes in basic biomedical research and in clinical diagnosis. However, the monoclonal antibodies produced in this manner are suitable for a number of applications in which monoclonal antibodies are needed in highly pure and concentrated form only after laborious further processing. In the in vivo production form (ascitic fluid), the monoclonal antibodies are already present in very high concentrations (up to 20 mg/ml) in a purity of 10% to 30% in the primary product. When monoclonal antibodies are produced with the usual stationary in vitro production methods, however, concentrations of only approximately 0.01 to 0.10 mg/ml in a purity of less than 1% (in most cases highly contaminated with animal serum proteins) are achieved.
To allow the production of monoclonal antibodies at higher concentration and higher purity in vitro as well, a method and an apparatus in the form of a culture vessel similar to a roller bottle have been proposed, in which a "supply chamber" with nutrients for the cells being supplied, and a plurality of "production chambers" arranged therein in which cell growth occurs and in which the monoclonal antibodies are produced, are separated from one another by semipermeable dialysis membranes. Cells are supplied with nutrients from the "supply chamber" through the semipermeable dialysis membrane, while waste products and metabolic products are discharged, again through the dialysis membrane, from the "production chambers" into the "supply chamber." This apparatus has become known as the "Bochum glass mouse." This culture vessel for cell cultures is described, for example, in the paper entitled "The Glassmouse: A Rollerbottle-like Apparatus for Culturing Hybridomas in Dialysis Bags," presented by T. Henglage, F. Haardt, and F. W. Falkenberg at the 1991 World Congress on Cell and Tissue Culture in Anaheim, Calif. on Jun. 16-20, 1991. This culture vessel for cell cultures consists of a glass tube with an outside diameter of 120 mm, the ends of which are turned outward to form flanges. The length of the glass tube including the flanges is 320 mm. The ends of the glass are sealed with 15-mm thick polymethyl methacrylate (PMMA) disks. One of the PMMA disks has 5 through holes, one of them along the long axis of the vessel and sealed with a stopper that in turn has two smaller openings which are used to admit a CO.sub.2 /air mixture and to equalize pressure. For this purpose, a stainless steel tube with an inside diameter of 1 mm is passed through one of the two openings and extends to the opposite end of the glass tube and the CO.sub.2 /air mixture is fed through this and a sterile filter into the interior of the vessel. The four remaining holes in the PMMA disk surrounding the central hole are used to introduce dialysis sacks, which project into the culture vessel and whose walls in each case consist of semipermeable dialysis membranes. The cell culture mixtures being cultured are placed in these dialysis sacks, which act as production chambers, while the interior of the culture vessel additionally serves as the supply chamber for the cells, and is filled with nutrient medium to approximately 40% of its volume. The cells are supplied with nutrients from the supply chamber through the semipermeable dialysis membranes, while waste products and metabolic products are also discharged through the dialysis membrane into the supply medium where they are diluted and neutralized. To allow the culture vessel to rotate about its long axis, it can be equipped with a sealed rotary lead through which the supply line for the CO.sub.2 /air mixture passes.
This apparatus, in which the cells enclosed in the production chambers are surrounded by the semipermeable dialysis membranes, allows hybridoma cells to be cultured over longer periods and at high densities (more than 10.sup.7 cells/ml). The known culture vessel, however, is a relatively complex apparatus which is difficult to handle, the construction of which requires a certain skill that not every laboratory shop can provide. In the known culture vessel, the gases necessary for cell culture metabolism and to create physiological conditions are supplied by introducing into the supply chamber the gas mixture which constitutes the surrounding atmosphere; oxygen physically dissolves in the nutrient medium, and is transported from there through the dialysis membrane into the production chamber. Although the transport of oxygen from the supply chamber through the dialysis membrane into the cell culture chamber is not very efficient, it is sufficient for cell densities up to about 10.sup.7 cells/ml. Higher cell densities require an improvement in oxygen supply. Since at higher cell densities the cells' oxygen requirement is so high that the oxygen content in the medium in cell culture chamber is exhausted in a few minutes, and the oxygen dissolved in the medium of the supply chamber is used up in less than one hour, additional oxygen must be delivered from the gas phase into the nutrient medium in the supply chamber by gas transfer. The weak point of the known culture vessel has proven to be the fact that continuous feeding of the CO.sub.2 /air mixture through the rotary lead through can cause infections of the cell cultures.
An object of the present invention therefore is to provide a culture vessel for the generation of cell cultures at high cell densities in which cell growth is not limited by an insufficient supply of oxygen to the cells, that can be produced economically and is easy to handle, and in which the danger of infections is reduced.